The possibility of using electrophoresis as a preparative separation technique has been investigated since the nineteen fifties.
In continuous free-flow electrophoresis, a solution flows into an electrophoresis chamber and is separated in the electrical field between two electrodes. On leaving the electrophoresis module, the liquid flow is divided into a large number of fractions which contain the substances to be separated at different concentrations. Although this technique leads to good selectivities on a laboratory scale, scaling-up is only possible within narrow limits. The main problem is considered to be the heating of the solution in the electrical field and the dispersion phenomena, such as heat convection, resulting from this. Productivities of only a few grams of product per day can be achieved using commercially available free-flow electrophoresis appliances.
In membrane electrophoresis, semipermeable membranes act as convection barriers between adjacent compartments, with at least one dissolved component being able to migrate, in the electrical field, from one compartment into another.
The first publications used macroporous membranes, for example filter paper. However, these materials suffer from a number of disadvantages: they do not exhibit any selectivity for the substances to be separated (U.S. Pat. No. 3,989,613, U.S. Pat. No. 4,043,895) and, in addition to this, frequently exhibit only a low degree of mechanical and chemical stability. Moreover, they are susceptible to non-negligible pressure-induced transmembrane flows at pressure differences of less than 1 kPa. Pressure differences, which can vary temporarily or locally, between individual compartments consequently lead to backmixing and to a decrease in separation efficiency. Gritzner reduced this pressure-induced permeate flow by linking to equilibration tanks (U.S. Pat. No. 4,043,895). By means of variable levels, the hydrostatic pressures in the two flow channels adjust themselves independently to the same value.
Subsequent patents propose using ultrafiltration membranes which exhibit selectivity for macromolecules of different sizes. In theory, the separation efficiency can consequently be markedly increased by combining the selectivity criteria, electrophoretic mobility and membrane retention. Aside from laid-open specifications without any implementation example (DE 3 337 669 A1, DE 3 626 953 A1), batch experiments on only a millilitre scale have been published (U.S. Pat. No. 6,270,672).
However, very low productivity is observed in practice when using ultrafiltration membranes. An electrical double layer develops in the membrane pores, leading, in the electrical field, to the induction of an electroosmotic flow which drastically reduces the capacity to separate negatively charged proteins (Galier et al., J. Membr. Sci. 194 [2001] 117-133).
If a protein species is positively charged under separation conditions, the separation can be carried out under reversed electrode polarity. In this case, the electroosmotic flow can be in the opposite orientation to the protein transport through the membrane or in the same orientation. Correspondingly, an increase or a decrease in the level of liquid is observed in the diluate container. While productivity decreases in the first case, the second case results in a decrease in separation efficiency since proteins of low mobility, which should remain in the diluate, can be transported convectively through the membrane. It is consequently not possible to equalize liquid flows through the separation membranes by equalizing hydrostatic pressure differences in the module, as described, for example, in U.S. Pat. No. 4,043,895.
The use of gel membranes represents an alternative to using porous ultrafiltration and microfiltration membranes. This nonporous material offers the advantage of low electroosmotic flow. The selectivity of the gel membrane for macromolecules can be influenced by the degree of polymer crosslinking (cf. U.S. Pat. No. 4,243,507).
However, gel membranes suffer from a number of disadvantages as compared with porous membranes:
They exhibit high resistance in an electrical field. This leads to high energy input and consequently to the evolution of a great deal of heat in the module.
Gels such polyacrylamide possess poor pH stability and cannot therefore be cleaned like conventional ultrafiltration and microfiltration membranes. This results in very high costs for module replacement since cleaning when handling proteins is essential.
On the one hand, the use of ultrafiltration and microfiltration membranes for membrane electrophoresis has thus far been limited by the electroosmotic effect. On the other hand, while gel membranes can be used, with the above-mentioned disadvantages, scaling-up fails because of the membrane costs and energy input being too high.
The invention is based on the object of developing an improved membrane electrophoresis method which uses ultrafiltration and microfiltration membranes and which does not suffer from the disadvantages mentioned here.
Because of the electroosmotic flow which develops in porous membranes, use of these membranes on an industrial scale (>0.5 kg/h), for example, has not previously been possible.